The present invention relates to recombinant DNA technology, and in particular to introducing foreign nucleic acid(s) into a eukaryotic host cell, and more particularly to producing infective, propagation-defective virus-like particles which collectively direct the expression of a representative set of immunogenic proteins (an expression library) of a pathogen (virus, fungus, bacterium or protozoan), parasite or tumor cell. These libraries have applications in human and veterinary medicine.
A vaccine is one of the most efficacious, safe and economical strategies for preventing disease and controlling the spread of disease. Conventional vaccines are a form of immunoprophylaxis given before disease occurrence to afford immunoprotection by generating a strong host immunological memory against a specific antigen. The primary aim of vaccination is to activate the adaptive specific immune response, primarily to generate B and T lymphocytes against specific antigen(s) associated with the disease or the disease agent.
Similarly, cancer vaccines aim to generate immune responses against cancer tumor-associated antigens. Cancers can be immunogenic and can activate host immune responses capable of controlling the disease and causing tumor regression. However, cancer at the same time can be specifically and nonspecifically immunosuppressive and can evade the host's immune system. Many protein/glycoprotein tumor-associated antigens have been identified and linked to certain types of cancer. Her-2-neu, PSA, PSMA, MAGE-3, MAGE-1, gp100, TRP-2, tyrosinase, MART-1, β-HCG, CEA, Ras; B-catenin, gp43, GAGE-1, BAGE-1, MUC-1,2,3, and HSP-70 are just a few examples.
Multiple approaches are being assessed in immunizing cancer patients with tumor-associated antigens (TAAs). Vaccines in clinical use fall into several categories determined by their components, which range from whole cells to immunogenic peptides. Whole cell and cell lysate vaccines can be autologous or allogeneic vaccines, depending on the host origin of the cancer cells. An autologous whole cell cancer vaccine is a patient-specific formulation made from the patient's own tumor. To date, many autologous cancer vaccines have not been clinically successful unless they are modified to increase their intrinsic immunogenicity, for example by the co-expression of lymphokines such as GM-CSF (Ward et. al., 2002. Cancer Immunol. Immunother. 51:351-7). Because they are patient-specific, they can also be costly and limited to those patients from whom cancer cells can be obtained in sufficient quantity to produce a single-cell suspension. In addition, the inherently limited number of cells is problematic with respect to the need for modification or for multiple vaccinations, making an autologous formulation impractical for prophylaxis or treatment of early disease. Some of these problems are solved with allogeneic whole cell vaccines or genetically engineered whole cell vaccines where instead of supplying immunostimulatory agents such as lymphokines exogenously with the tumor vaccine, the tumor cells are genetically modified to express the lymphokine endogenously. However, these methods may be time consuming and prohibitively expensive to produce.
Natural and recombinant cancer protein antigen vaccines are subunit vaccines. Unlike whole cell vaccines, these subunit vaccines contain defined immunogenic antigens at standardized levels. The key problem with such vaccines is finding the right adjuvant and delivery system. In addition, purification of natural or recombinant tumor antigens is tedious and not always logistically practical. Protein cancer vaccines require culturing tumor cells, purifying tumor antigens, or producing specific peptides or recombinant proteins. In addition, vaccines that are made solely from tumor protein/peptides pose intrinsic problems in that they can be limited in the ability to be directed into the correct antigen presentation pathways or may not be recognized by the host due to host major histocompatibility complex (MHC) polymorphisms. For these reasons, whole cell, or vector delivered tumor vaccines expressing a large array of tumor antigens are anticipated to be preferred vaccination methods. Vaccines which include nucleic acid encoding the tumor antigens rather than vaccines comprising the antigen itself, address some of these problems. To date these approaches have shown the most promise in pre-clinical and clinical testing. Amongst the current technologies being applied to cancer vaccination, two particular systems have shown significant potential for application in this field. The first is delivery of TAAs using viral vectors, including but not limited to adenoviral, adeno associated virus, retroviral, poxviruses, flaviviruses, picornaviruses, herpesviruses and alphaviruses (see WO 99/51263). The second is vaccination with tumor cell protein or RNA using ex vivo derived dendritic cells as the delivery vehicle for transfer and expression of the TAAs into the host (Heiser et al., 2002. J. Clin. Inv. 109:409-417 and Kumamoto et al., 2002. Nature Biotech. 20:64-69).
A limiting factor in many tumor vaccine approaches appears to be the limited availability of known tumor-specific antigens. These tumor-specific antigens can vary not only between tissue type from which the tumor originated, but may even vary from cell-to-cell within the same tumor. A confounding problem associated with using only a limited number of tumor antigen targets in a vaccine is the potential for “tumor escape” where the tumor essentially evades detection by the vaccine induced immune effector cells by deleting certain tumor associated antigens.
This observation prompted investigators to design cancer vaccines expressing multiple antigens to reduce the propensity of tumor escape. Unfortunately due to the limited number of antigens that have been identified to date, this is not a feasible approach for the majority of tumors. Therefore, a more recent evolution of cancer therapy has been the use of entire tumor antigen libraries. This combines multiple beneficial characteristics one would want in a cancer vaccine. A vaccine encoding an entire tumor antigen repertoire negates the need for antigen identification and isolation; essentially the vaccine recipient's immune system is allowed to make this choice in determining which TAAs the individual will respond to. The second distinct advantage of this approach is that, since the repertoire of antigens being expressed is so broad, the chance of tumor escape is minimized or eliminated entirely. Currently this approach is most actively being pursued using dendritic cells to deliver tumor antigen libraries. These cells, which function as antigen presenting cells by presenting the tumor antigens to the immune system, are isolated from each cancer patient, cultured and expanded in vitro, loaded with tumor antigen either in the form of protein or nucleic acid; see U.S. Pat. Nos. 5,853,719 and 6,306,388. This approach has generated promising clinical data in human testing and has shown the ability to retard tumor growth in some individuals, and even to drive tumor regression in a number of patients (Sadanaga et. al., 2001, Clin. Cancer Res. 7:2277-84). The major drawback for this technology is the need for in vitro culture, expansion and antigen loading of the patient derived dendritic cells prior to vaccination of each individual. This is a time consuming and expensive process, and can be highly variable since the dendritic cell population from individual to individual can vary widely in its phenotype, growth characteristics and activity.
To date, naked DNA, RNA, viral and bacterial vectors have been tested for their ability to induce cancer specific responses against a tumor antigen library. An alternative approach is the use of viral vectors to deliver a tumor antigen library to a cancer patient. To date, some success has been achieved with naked nucleic acid expression libraries; e.g., see U.S. Pat. Nos. 5,989,553 and 5,703,057. Attempts to augment the immune responses elicited to naked nucleic acid vectors include the use of self-replicating viral vectors delivered in the form of naked RNA or DNA (Ying et al., 1999, Nature Medicine, 5:823-827).
Viral vectors have shown great promise in pre-clinical and clinical testing for prevention of a number of infectious disease targets. One of the most pressing issues for development of viral vectors for prophylactic and therapeutic vaccine uses in humans is the ability to produce enough particles in a regulatory acceptable form. For many viral systems, this goal is within reach and a number of vector systems have produced positive immune response and safety profiles in clinical trials. However, most production schemes for vaccine vector platforms are focused on production of large quantities of vaccine particles expressing single or at the most two or three known antigens for specific disease targets e.g. the gag, pol and env genes of HIV in poxvirus vectors. However, in most cases, these large-scale manufacturing approaches are not practical for the manufacture of individual patient-specific vaccines.
Alphaviral vector delivery systems have been identified as attractive vaccine vectors for a number of reasons including: high expression of heterologous gene sequences, the derivation of non-replicating (alpha)virus replicon particles (ARP) with good safety profiles, an RNA genome which replicates in the cytoplasm of the target cell and negates the chance of genomic integration of the vector, and finally the demonstration that certain alphaviral vectors are intrinsically targeted for replication in dendritic cells and thus can generate strong and comprehensive immune responses to a multitude of vaccine antigens (reviewed in Rayner, Dryga and Kamrud, 2002, Rev. Med. Virol. 12:279-296). The Alphavirus genus includes a variety of viruses, all of which are members of the Togaviridae family. The alphaviruses include Eastern Equine Encephalitis Virus (EEE), Venezuelan Equine Encephalitis Virus (VEE), Everglades Virus, Mucambo Virus, Pixuna Virus, Western Equine Encephalitis Virus (WEE), Sindbis Virus, Semliki Forest Virus, Middleburg Virus, Chikungunya Virus, O'nyong-nyong Virus, Ross River Virus, Barmah Forest Virus, Getah Virus, Sagiyama Virus, Bebaru Virus, Mayaro Virus, Una Virus, Aura Virus, Whataroa Virus, Babanki Virus, Kyzylagach Virus, Highlands J Virus, Fort Morgan Virus, Ndumu Virus, and Buggy Creek Virus. The viral genome is a single-stranded, messenger-sense RNA, modified at the 5′-end with a methylated cap and at the 3′-end with a variable-length poly (A) tract. Structural subunits containing a single viral protein, C, associated with the RNA genome in an icosahedral nucleocapsid. In the virion, the capsid is surrounded by a lipid envelope covered with a regular array of transmembrane protein spikes, each of which consists of a heterodimeric complex of two glycoproteins, usually E1 and E2. See Pedersen et al., J. Virol 14:40 (1974). The Sindbis and Semliki Forest viruses are considered the prototypical alphaviruses and have been studied extensively. See Schlesinger, The Togaviridae and Flaviviridae, Plenum Publishing Corp., New York (1986). The VEE virus has also been extensively studied. See, e.g., U.S. Pat. No. 5,185,440, and other references cited herein.
The studies of these viruses have led to the development of techniques for vaccination against the alphavirus diseases and against other diseases through the use of alphavirus vectors for the introduction of foreign DNA encoding antigens of interest. See U.S. Pat. No. 5,185,440 to Davis et al., and PCT Publication WO 92/10578. The introduction of foreign expressible DNA into eukaryotic cells has become a topic of increasing interest. It is well known that live, attenuated viral vaccines are among the most successful means of controlling viral disease. However, for some viral (or other) pathogens, immunization with a live virus strain may be either impractical or unsafe. One alternative strategy is the insertion of sequences encoding immunizing antigens of such agents into a live, replicating strain of another virus. One such system utilizing a live VEE vector is described in U.S. Pat. No. 5,505,947 to Johnston et al. Another such system is described by Hahn et al., 1992, Proc. Natl. Acad. Sci. USA 89:2679-2683, wherein Sindbis virus constructs express a truncated form of the influenza hemagglutinin protein. Another approach is the use of infective, propagation-defective alphavirus particles, as described in U.S. Pat. No. 6,190,666 to Garoff et al., U.S. Pat. Nos. 5,792,462 and 6,156,558 to Johnston et al., U.S. Published Application No. 2002/0015945 A1 (Polo et al.), U.S. Published Application No. 2001/0016199 (Johnston et al.), Frolov et al., 1996, Proc. Natl. Acad. Sci. USA 93:11371-11377 and Pushko et al. (1997) Virology 239:389-401. Alphaviruses have also been shown to be relatively easy to genetically manipulate, as reflected by a number of applications using alphaviruses as genomic expression libraries, e.g., see U.S. Pat. No. 6,197,502. The use of Semliki Forest Virus (SFV) vectors expressing a library of antigens has also been explored in animal models where SFV particles expressing a library of tumor antigens were used to infect dendritic cells in vitro and the dendritic cells were used to immunize mice showing some protection in a glioma model (Yamanaka et al., 2001, J. Neurosurg. 94:474-81).
There is a longfelt need in the art for nucleic acid sequences encoding foreign antigens which can be used to immunize a person or an animal against neoplastic conditions or against parasite or pathogen infection, especially where there is no attenuated strain or where the neoplasia, parasite or pathogen is not well characterized at the molecular level, or where it is recognized that protective immunization requires the expression of multiple antigens.